title: Determination of CK2 specificity and substrates by proteome-derived peptide libraries.
abstract: Understanding the specificity of kinases enables prediction of their substrates and uncovering kinase functions in signaling pathways. Traditionally synthesized peptide libraries are used to determine the kinase specificity. In this study, a proteomics-based method was developed to determine the specificity of kinase by taking the advantages of proteome-derived peptide libraries and quantitative proteomics. Proteome-derived peptide libraries were constructed by digesting proteins in total cell lysate followed with dephosphorylation of the resulting peptides. After incubating the peptide libraries with/without CK2 for in vitro kinase assay, stable isotopic labeling based quantitative phosphoproteomics was applied to distinguish the in vitro phosphosites generated by CK2. By using the above approach, 404 CK2 in vitro phosphosites were identified by 1D LC-MS/MS. Those sites allowed the statistic determination of the CK2 specificity. In addition to the easy construction of the proteome-derived peptide library, another significant advantage of this method over the method with synthesized peptide libraries is that the identified phosphosites could be directly mapped to proteins for the screening of putative kinase substrates. It was found that the confidence for substrate identification could be significantly improved by comparing the in vitro CK2 sites with the in vivo sites identified by phosphoproteomics analysis of the same cell lines. By applying this integrated strategy, 138 phosphosites from 105 putative CK2 substrates of high confidence were determined.
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Chunli
authors.initials: C
authors.lastname: Wang
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Bian
authors.forename: Fangjie
authors.initials: F
authors.lastname: Liu
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.forename: Mingming
authors.initials: M
authors.lastname: Dong
authors.forename: Jing
authors.initials: J
authors.lastname: Dong
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D000595:Amino Acid Sequence; D047390:Casein Kinase II / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis*; D019151:Peptide Library; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D010446:Peptide Fragments; D019151:Peptide Library; D010750:Phosphoproteins; D047390:Casein Kinase II